We have used a unique cell culture model to study in vitro catabolism of high density lipoproteins (HDL) and to identify cubilin as a receptor that mediates endocytosis and lysosomal degradation of holoparticle HDL. The activities of cubilin are distinct from those of the other known HDL receptor, scavenger receptor SR-BI, which mediates selective lipid uptake without facilitating endocytosis and degradation of the whole particle. Cubilin is a 460-kDa peripheral membrane protein that is expressed in kidney, intestine and yolk sac edoderm. The physiological significance of cubilin activity with respect to HDL endocytosis in these tissues is unknown. The vital role served by yolk sac cubilin is indicated by the severe developmental abnormalities observed when cubilin antibodies are administered to pregnant rats or to embryos cultured ex utero. A central hypothesis of this grant application is that cubilin functions in the yolk sac endoderm to mediate uptake of maternal plasma HDL and thereby serves a role supplying the embryo with HDL-associated cholesterol, lipid-soluble vitamins, phospholipids and/or triglycerides. To test this hypothesis, and to characterize fundamental mechanistic features of cubilin function, four aims are proposed. The first aim is to define the molecular basis for ligand binding and endocytotic functions of cubilin and will include mapping binding sites for HDL apolipoproteins A-I and A-II, and establishing the basis for the association of cubilin with the plasma membrane as it lacks a membrane-spanning domain. To accomplish these goals, recombinant fragments of cubilin and monoclonal antibodies will be produced and tested using in vitro binding and cellular uptake assays. The second aim is to determine whether other cell surface proteins function in conjunction with cubilin to mediate HDL endocytosis. These studies will evaluate megalin, a receptor that binds cubilin, and several other proteins that copurify with cubilin in the process of cublilin-mediated HDL endocytosis. The third aim is to test the hypothesis that cubilin not only functions to mediate lysosomal degradation of HDL but also mediates transcytosis as a means to transport HDL or its constituents across certain epithelia. This study will involve measuring transport of radiolabeled HDL across a monolayer of cubilin-expressing cells cultured on a permeable support. The fourth aim is to determine the developmental significance of cubilin-mediated uptake of HDL by yolk sac endoderm. Together, these studies should provide new insights into the function of cubilin in processes such as maternal-embryonic lipoprotein transport and embryonic development.